Equine protozoal myeloencephalitis (EPM) is a treatable, but often fatal, central nervous system (CNS) disease of equids. It has not been reported among horses originating outside the Western hemisphere. Several hundred cases occur in North America annually. Although EPM has been reported in ponies, donkeys and most breeds of horses, the greatest incidence has been among thoroughbreds, standardbreds, and quarter horses..sup.1 (See references section below.) The disease occurs as a result of infection with Sarcocystis neurona. Merozoites multiply in neurons, leucocytes and vascular endothelial cells of the CNS resulting in perivascular mononuclear cell infiltration and necrosis of the neuropile. Antemortem diagnosis of EPM is difficult..sup.2 Clinical signs vary dramatically, depending upon the location and severity of CNS lesions. The disease may mimic various neurological disorders of the horse. Clinicopathologic data are frequently of little diagnostic value.
Some methods of diagnosing equine parasitic and other infections are known. For example, U.S. Pat. No. 4,740,456 to Kuhn et al. discloses immunological methods for diagnosing active human neurocysticercosis, including a serum test and a cerebrospinal fluid test. The test involves detecting an antigen or antigens of larval origin, specifically of Taenia solium larva.
U.S. Pat. No. 4,759,927 to Dutta discloses a vaccine against Potomac Horse Fever comprising deactivated E. Risticii as the active agent. The patent also discloses an assay for detecting the presence of E. Risticii antibodies.
U.S. Pat. No. 5,141,925 to Alroy et al. discloses a method for the prophylactic and therapeutic treatment of animals having a parasite able to cause Coccidiosis.
U.S. Pat. No. 5,210,018 to Nuzzolo et al. discloses an immunoenzymatic method for the detection of anti-Plasmodium falciparum-sporozoite antibodies in human blood.
"Evidence for Sarcocystis as the etiologic agent of equine protozoal myeloencephalitis", Simpson et al., J Protozool (UNITED STATES) August 1980, 27 (3) p288-92, discloses the diagnosis of Equine protozoal myeloencephalitis (EPM) in 10 horses. By electron microscopy, schizonts were found in intact host cells of the spinal cords or, more frequently, free in the extracellular spaces. Developmental stages of schizonts differed morphologically, and the late stage of schizogony was characterized by endopolygeny. These findings permitted tentative identification of the protozoan as a Sarcocystis sp. Free merozoites were present in the extracellular spaces or in cells of the spinal cord. Pericytes of capillaries were most frequently parasitized by merozoites, but the cytoplasm of neurons, macrophages, intravascular and tissue neutrophils, and axons of myelinated nerve fibers also contained these organisms. The presence of parasites in the cytoplasm of tissue and circulating neutrophils suggest that this putative Sarcocystis sp. may have a hematogenous phase of infection.
"Immunohistochemical study to demonstrate Sarcocystis neurona in equine protozoal myeloencephalitis", Hamir et al., J. Vet. Diagn. Invest. (UNITED STATES) July 1993, 5 (3) p418-22; discloses a 5-year (1985-1989) retrospective immunohistochemical study was conducted using an avidin-biotin complex (ABC) immunoperoxidase method to demonstrate Sarcocystis neurona in histologically suspect cases of equine protozoal myeloencephalitis (EPM). Primary antibodies against S. neurona and S. cruzi were utilized for the ABC technique. The findings were compared with those from cases in which the organisms were detected by examination of hematoxylin and eosin (HE)-stained neuronal sections. HE-stained sections detected the presence of the organisms in 20% of the suspect cases; whereas the ABC technique confirmed the presence of S. neurona in 51% and 67% of the cases by S. neurona and S. cruzi antibodies, respectively. A review of clinical case histories showed that 21/47 (45%) of the EPM horses with parasites in the tissue sections had prior treatment with antiprotozoal drugs and/or steroids. Using the test results of S. neurona and S. cruzi as a standard reference, HE test sensitivity based on examination of up to 30 neuronal sections per case was only 25%, and test specificity was 91%.
"Equine protozoal myeloencephalitis: Antigen analysis of cultured Sarcocystis neurona merozoites", Granstrom et al., J. Vet. Diagn. Invest. (UNITED STATES) January 1993, 5(1) p 88-90; discloses antigens of cultured Sarcocystis neurona merozoites were examined using immunoblot analysis. Blotted proteins were probed with S. cruzi, S. muris, and S. neurona antisera produced in rabbits, S. fayeri (pre- and post-infection) and S. neurona (pre- and post- inoculation) sera produced in horses, immune sera from 7 histologically confirmed cases of equine protozoal myeloencephalitis (EPM), and pre-suckle serum from a newborn foal. Eight proteins, 70, 24, 23.5, 22.5, 13, 11, 10.5, and 10 Kd, were detected only by S. neurona antiserum and/or immune serum from EPM-affected horses. Equine sera were titered by the indirect immunofluorescent antibody (IFA) method using air-dried, cultured S. neurona merozoites. Anti-Sarcocystis IFA titers were found in horses with or without EPM. Serum titers did not correspond to the number of specific bands recognized on immunoblots.
"A five year (1985-1989) retrospective study of equine neurological diseases with special reference to rabies", Hamir et al., J. Comp. Pathol. (ENGLAND) May 1992, 106 (4) p411-21; discloses a retrospective study of horses necropsied between 1985 and 1989 at a diagnostic laboratory of a veterinary school in North America is documented. In this investigation over 20 per cent of the horses had clinical neurological signs. Equine protozoal myeloencephalitis (caused by Sarcocystis neurona) and cervical stenotic myelopathy (wobbler syndrome) were the most common of these disorders. However, only four cases of equine rabies were diagnosed during the 5-year study. The gross microscopical and immunohistochemical findings from these rabies-positive horses are documented. Immunoperoxidase tests for detection of rabies antigen in another 35 horses with non-specific encephalitis/encephalopathydid not reveal any positive cases. Based on this investigation, it appears that immunoperoxidase is a valid method for diagnosis of rabies when fresh tissues are not available for the fluorescent antibody test.
"Characterization of Sarcocystis neurona from a thoroughbred with equine protozoal myeloencephalitis", Bowman et al., appears in Cornell Vet. 1992, April 82(2):115, Cornell Vet. (UNITED STATES) January 1992, 82 (1) p41-52 and discloses morphological information for syntype material of the etiologic agent of equine protozoal myeloencephalitis, Sarcocystis neurona. A clinical description of the horse from which the organism was isolated and the methodology used to immunosuppress the horse in an attempt to increase parasite numbers are also given. The description includes microscopic details observed both with light and transmission electron microscopy. Mainly stages from tissue are illustrated, but information is also presented on the development of the organism after inoculation on to monolayers of bovine monocytes. It is believed that the large numbers of organisms observed in this horse were due to its having not received prior treatment with trimethoprimsulphonamide and the large amounts of corticosteroids that were administered in order to facilitate isolation of the pathogen.
"Sarcocystis neurona n. sp. (Protozoa: Apicomplexa), the etiologic agent of equine protozoal myeloencephalitis", Dubey et al., J Parasitol (UNITED STATES) April 1991, 77 (2) p212-8; discloses Sarcocystis neurona n. sp. is proposed for the apicomplexan taxon associated with myeloencephalitis in horses. Only asexual stages of this parasite presently are known, and they are found within neuronal cells and leukocytes of the brain and spinal cord. The parasite is located in the host cell cytoplasm, does not have a parasitophorous vacuole, and divides by endopolygeny. Schizonts are 5-35 microns .times.5-20 microns and contain 4-40 merozoites arranged in a rosette around a prominent residual body. Merozoites are approximately 4.times.1 micron, have a central nucleus, and lack rhoptries. Schizonts and merozoites react with Sarcocystis cruzi antiserum but not with Caryospora bigenetica. Toxoplasma gondii, Hammondia hammondi, or Neospora caninum antisera in an immunohistochemical test.
"Equine protozoal myeloencephalitis", Madigan et al., Vet. Clin. North Am. Equine Pract. (UNITED STATES) August 1987, 3 (2) p397-403; discloses Equine protozoal myeloencephalitis (EPM) is a disease that produces neurologic signs of brain or spinal cord dysfunction. The causative organism is believed to be a Sarcocystis species of protozoa. A definitive diagnosis can only be made on histopathology of affected spinal cord or brain. No preventive measures or documented treatment is available at this time for suspected cases of EPM.
Currently the only method of antemortem diagnosis of equine sarcocystis (EPM) is by Western Blot, which detects the presence of antibodies against S. neurona. Blood contamination of the spinal fluid, or loss of integrity of the blood-brain barrier may result in false positive tests. A DNA based test according to the present invention circumvents these problems. Recently, progress has been made in the development of serological methods for EPM diagnosis. The availability of cultured merozoites has made it possible to detect S. neurona-specific antibodies in serum and cerebrospinal fluid of affected horses..sup.3 Immunoblot analysis of these samples distinguishes between exposure to S. neurona and S. fayeri a common, non-pathogenic, coccidia of the horse. Previous serologic tests relied on S. cruzi bradyzoite antigen which only detected antibodies to shared or crossreactive Sarcocystis spp. epitopes. Preliminary immunoblot screening of several groups of horses in central Kentucky indicated that many horses are exposed while few develop clinical signs ..sup.4 At present, there is no method to directly detect and differentiate S. neurona from non-pathogenic Sarcocystis in live animals.
There is a need in the art for a diagnostic method of detecting Equine protozoal myeloencephalitis (EPM). The primer/probe of the present invention provides the first diagnostic primer for the diagnosis of Equine protozoal myeloencephalitis (EPM) caused by the parasite Sarcocysitis neurona.